Methods and devices of this kind are used, for example, in biomedical research, active ingredient research, biomaterial research or toxicology to analyze the activation of a target cell by an activator. Such studies are thus investigating whether activation is actually identifiable in a specific case and optionally also the aspect of more detailed characteristics of the activation, if activation can indeed be identified.
Many processes in the body of organisms are triggered by a cell, also referred to as a “target cell”, coming into contact with a particle, with an extended solid or with another cell. The cell is activated by the contact. This means that contact between the cell and the particle, the solid or the other cell triggers cell-internal signals, then leading, for example, to a modified gene expression pattern of the cell. The cell is thereby converted into a new state and fulfils new functions.
One example of cell activation is the action of one or more adjuvants on the target cell. An adjuvant is often defined as an auxiliary substance which enhances the action, on the target cell, of substances having a specific action. In immunology an adjuvant is, for example, required in order to obtain an adequate immune response. Different adjuvants may also improve the immune response in a different manner. Where a vaccination is given, an adjuvant is, for example, also administered in order to ensure adequate protection by vaccination.
The exact mode of action of adjuvants is often still unknown, and their action is established empirically. In Europe at present only a very small number of adjuvants is approved for vaccination. For the discovery of an improved or new adjuvant, at present in immunology, for example, the immune response has to be analyzed at least in part. One of the ways of analyzing the immune response is to analyze the activation state of immune cells, for example of dendritic cells. This usually requires an appropriate antibody, which also has to be labeled with a fluorophore. An incubation period, which can easily last several hours, also has to be waited out. To achieve adequate statistics a sufficient number of cells is also required. In many cases an animal experiment will also be unavoidable if the action of the adjuvant is to be evaluated.
It is known that, for example, adjuvants cause activation or at least enhanced activation of immune cells. There are numerous further examples in immunology where, in very general terms, an activator activates a target cell which then, according to the activation, exhibits different behavior, at least in part, from before the activation. Thus, for example, T cells are activated only by dendritic cells and release cytokines, for example. B cells are activated by antigens and secrete corresponding antibodies. The generalized significance of particle-based cell activation is made clear by the mode of action of an active ingredient particle. An active ingredient particle becomes active only when, on contact, it activates a target cell.
A further example of particle-based cell activation is the action of a toxic particle. It is known that particles can develop a noxious action, for example when inhaled into the lung. The noxious action is produced only when the particle, on contact, activates a cell, for example a pulmonary epithelial cell.
One example of solid-based cell activation can be found in transplant technology. If, for example, a transplant material coming into contact with cells of a tissue leads to activation of the cells, this can lead to inflammation of the tissue, and the transplant is rejected. Generally speaking, in biomaterial research it must be ensured that the desirable cell activation occurs and undesirable cell activation is excluded.
Cell activation resulting from contact with other cells occurs, for example, in T cell selection and activation (Alberts et al., “Molekularbiologie der Zelle”, VCH-Verlag, Weinheim, 1995), in the migration of neutrophils (Wagner et al., “Neutrophil Migration Mechanisms, with an Emphasis on the Pulmonary Vasculature”, Pharmacological Reviews, Vol. 52, Issue 3, 349-374, 2000), in organogenesis generally or in processes connected with the formation of a cancerous ulcer or the formation of secondary cancers.
Probe microscopy (PM) is a measuring and analytical technique where, in a prominent design, a measuring probe is raster scanned over a sample of a measuring medium that is to be analyzed and where a topography of the sample is established by means of a distance-dependent interaction between the measuring probe and the sample. However, material constants or other sample information can also be obtained. Owing to the frequent use of a scanning process, the term scanning probe microscopy (SPM) is also often used instead of PM. There are different probe designs. The most prominent examples of PM are the atomic force microscope (AFM) and the scanning tunneling microscope (STM). Further examples of this technology are, in particular, the scanning near-field microscope (SNOM), the scanning photonice force microscope (SPhM) and the photonic force microscope. One further important analyzing method associated with probe microscopy is distance spectroscopy; here, for measurement purposes, the measuring probe is displaced usually only in a vertical direction relative to the sample analyzed. In this case both the instrument and the associated software can be reduced thereto.
To measure the distance-dependent interaction between the measuring probe and the sample, in distance spectroscopy the measuring probe is displaced relative to the surface of the sample, for example in a direction vertical to the sample surface, and the interaction between the measuring probe and the sample is measured. Alternatively the sample can also be moved. It is also possible to provide a relative movement between the measuring probe and the sample, where both the measuring probe and the sample are moved. In probe microscopy, this distance spectroscopy for measuring the interaction between the measuring probe and the sample is, for example, used to measure forces between molecules as one molecule binds to the measuring probe and a further molecule binds to the sample. The interaction between the two bound molecules can then be measured. It is, however, also possible to measure intramolecular forces, for example, by lowering the measuring probe onto the sample and binding is thus awaited. Thereafter the measuring probe can be removed from the sample again; during this process, forces thus acting on the measuring probe are recorded. Also possible are further measurements involving the measurement of an interaction which correlates with an associated distance between two or more locations.
In atomic force microscopy the measuring probe used is usually a component which is also referred to as a “cantilever”. This can be used to measure forces by recording the deflection of the measuring probe. To minimize the interaction space and thus to improve lateral resolution, in many cases a measuring tip is attached to the free end of the cantilever. In the case of distance spectroscopy, however, instead of a tip being provided the cantilever is also, for example, specifically loaded with a cell; often the tip is then even dispensed with. Without loss of generality, reference is made in the explanations below to a cantilever. The statements apply accordingly to other forms of measuring probes in probe microscopy. Cantilevers are usually secured to a base, in particular to ensure reasonable handling.
It is known to use both untreated and also pre-treated cantilevers as measuring probes for distance spectroscopy. In the case of an untreated cantilever, binding of the sample during measurement is non-specific. For example, this involves pulling molecules from their surrounding medium by means of binding to the cantilever in order to measure the interaction of the molecules with the surrounding medium. However, this also makes it possible to characterize more precisely the molecules being pulled. Thus, for example, DNA molecules exhibit a specific spectroscopy curve on the basis of an internal conformational change.
A pre-treated cantilever enables an analysis to be made of, in particular, specific bonds. An analysis of this kind can be advantageous if the formation of undesirable bonds, which may possibly thereafter be virtually indistinguishable from each other, is to be prevented during measurement. Thus it is common practice to bind one or more molecules to the cantilever-type measuring probe, which then forms a receptor-ligand system with the bound molecule or molecules. It is also known to bind whole cells to a cantilever-type measuring probe and to bring this system into interaction with a sample, for example a biomaterial, or with other cells. In this case it may, for example, as already mentioned above, be beneficial to use a cantilever without a tip. Different forms of pre-treatment are known for measuring probes, in particular for cantilevers, for example that of making the measuring probe water-repellent.
Known possibilities for pre-treatment of the cantilever generally lead to coating of the measuring probe, at least sectionally. Thus a cell attached to the cantilever coats a section of the surface of the cantilever. In this case it is possible for the cantilever, in the course of the pretreatment, to be initially provided with a coating, in particular an adhesion-promoting coating, to which a substance to be measured is then applied. The material applied to the measuring probe, in particular the cantilever, in the course of the pretreatment is generally referred to hereafter as “probe substance”, whether this be an individual material or a combination of a plurality of materials comprising, for example, an adhesion-promoting base and a substance arranged thereon and to be analyzed. A (base) coating applied in the course of the pretreatment and encompassed by the probe substance is also referred to as “probe coating”.